RESUMO
OBJECTIVE: Glucocorticoid resistance is a rare endocrine disease caused by variants of the NR3C1 gene encoding the glucocorticoid receptor (GR). We identified a novel heterozygous variant (GRR569Q) in a patient with uncommon reversible glucocorticoid resistance syndrome. METHODS: We performed ex vivo functional characterization of the variant in patient fibroblasts and in vitro through transient transfection in undifferentiated HEK 293T cells to assess transcriptional activity, affinity, and nuclear translocation. We studied the impact of the variant on the tertiary structure of the ligand-binding domain through 3D modeling. RESULTS: The patient presented initially with an adrenal adenoma with mild autonomous cortisol secretion and undetectable adrenocorticotropin hormone (ACTH) levels. Six months after surgery, biological investigations showed elevated cortisol and ACTH (urinary free cortisol 114â µg/24â h, ACTH 10.9â pmol/L) without clinical symptoms, evoking glucocorticoid resistance syndrome. Functional characterization of the GRR569Q showed decreased expression of target genes (in response to 100â nM cortisol: SGK1 control +97% vs patient +20%, P < .0001) and impaired nuclear translocation in patient fibroblasts compared to control. Similar observations were made in transiently transfected cells, but higher cortisol concentrations overcame glucocorticoid resistance. GRR569Q showed lower ligand affinity (Kd GRWT: 1.73â nM vs GRR569Q: 4.61â nM). Tertiary structure modeling suggested a loss of hydrogen bonds between H3 and the H1-H3 loop. CONCLUSION: This is the first description of a reversible glucocorticoid resistance syndrome with effective negative feedback on corticotroph cells regarding increased plasma cortisol concentrations due to the development of mild autonomous cortisol secretion.
Assuntos
Glucocorticoides , Erros Inatos do Metabolismo , Receptores de Glucocorticoides , Humanos , Hormônio Adrenocorticotrópico/genética , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Glucocorticoides/metabolismo , Hidrocortisona , Ligantes , Mutação , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/deficiência , SíndromeRESUMO
Eukaryotic initiation factor 2 (eIF2) plays a key role in protein synthesis and in its regulation. The assembly of this heterotrimeric factor is facilitated by Cdc123, a member of the ATP grasp family that binds the γ subunit of eIF2. Notably, some mutations related to MEHMO syndrome, an X-linked intellectual disability, affect Cdc123-mediated eIF2 assembly. The mechanism of action of Cdc123 is unclear and structural information for the human protein is awaited. Here, the crystallographic structure of human Cdc123 (Hs-Cdc123) bound to domain 3 of human eIF2γ (Hs-eIF2γD3) was determined. The structure shows that the domain 3 of eIF2γ is bound to domain 1 of Cdc123. In addition, the long C-terminal region of Hs-Cdc123 provides a link between the ATP and Hs-eIF2γD3 binding sites. A thermal shift assay shows that ATP is tightly bound to Cdc123 whereas the affinity of ADP is much smaller. Yeast cell viability experiments, western blot analysis and two-hybrid assays show that ATP is important for the function of Hs-Cdc123 in eIF2 assembly. These data and recent findings allow us to propose a refined model to explain the mechanism of action of Cdc123 in eIF2 assembly.
Assuntos
Deficiência Intelectual Ligada ao Cromossomo X , Proteínas de Saccharomyces cerevisiae , Humanos , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/genética , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Aldosterone, the main mineralocorticoid hormone in humans, plays a pivotal role in the control of water and salt reabsorption via activation of the mineralocorticoid receptor (MR). Alterations in MR signaling pathway lead to renal dysfunction, including chronic kidney disease and renal fibrosis, that can be prevented or treated with mineralocorticoid receptor antagonists (MRAs). Here, we used RNA-Sequencing to analyze effects of two MRAs, spironolactone and finerenone, on the aldosterone-induced transcriptome of a human renal cell line stably expressing the MR. Bioinformatics analysis of the data set reveals the identity of hundreds of genes induced or repressed by aldosterone. Their regulation is modulated in a time-dependent manner and, for the induced genes, depends on the aldosterone-driven direct binding of the MR onto its genomic targets that we have previously characterized. Although both MRAs block aldosterone-induced as well as aldosterone-repressed genes qualitatively similarly, finerenone has a quantitatively more efficient antagonism on some aldosterone-induced genes. Our data provide the first complete transcriptome for aldosterone on a human renal cell line and identifies pro-inflammatory markers (IL6, IL11, CCL7, and CXCL8) as aldosterone-repressed genes.
Assuntos
Aldosterona/farmacologia , Rim/metabolismo , Naftiridinas/farmacologia , Espironolactona/farmacologia , Imunoprecipitação da Cromatina , Humanos , Rim/efeitos dos fármacos , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Transcriptoma/genéticaRESUMO
21-Hydroxylase deficiency (21OHD) is a rare genetic disorder in which salt-wasting syndrome occurs in 75% of cases, due to inability to synthesize cortisol and aldosterone. Recent mass spectrometry progress allowed identification of 21-deoxysteroids, i.e., 17-hydroxyprogesterone (17OHP), 21-deoxycortisol (21DF), and 21-deoxycorticosterone (21DB). We hypothesized that they may interfere with mineralocorticoid signaling and fludrocortisone therapy in patients with congenital adrenal hyperplasia (CAH) without effective glucocorticoid replacement and ACTH suppression. Our goal was to quantify circulating 21-deoxysteroids in a pediatric cohort with CAH related to 21OHD and to examine their impact on mineralocorticoid receptor (MR) activation. Twenty-nine patients with salt-wasting phenotype were classified in two groups according to their therapeutic control. During routine follow-up, 17OHP, 21DF, 21DB, and cortisol levels were quantified by liquid chromatography with tandem mass spectrometry before hydrocortisone intake and 1 and 2.5 h following treatment administration. Luciferase reporter gene assays were performed on transfected HEK293T cells while in silico modeling examined structural interactions between these steroids within ligand-binding domain of MR. Plasma 17OHP, 21DF, and 21DB accumulate in uncontrolled patients reaching micromolar concentrations even after hydrocortisone intake. 21DF and 21DB act as partial MR agonists with antagonist features similar to 17OHP, consistent with altered anchoring to Asn770 and unfavorable contact with Ala773 in ligand-binding pocket of MR. Our results demonstrate a complex interaction between all accumulating 21-deoxysteroids in uncontrolled 21OHD patients and mineralocorticoid signaling and suggest that appropriate steroid profiling should optimize management and follow-up of such patients, as keeping those steroids to low plasma levels should attest therapeutic efficacy and prevent interference with MR signaling.
Assuntos
Hiperplasia Suprarrenal Congênita/fisiopatologia , Mineralocorticoides , Transdução de Sinais , Esteroides/metabolismo , 17-alfa-Hidroxiprogesterona/sangue , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Cortodoxona/sangue , Feminino , Células HEK293 , Humanos , Hidrocortisona/metabolismo , Lactente , Masculino , Simulação de Acoplamento Molecular , Receptores de Mineralocorticoides/agonistas , Receptores de Mineralocorticoides/metabolismo , Adulto JovemRESUMO
PURPOSE: Primary ovarian insufficiency (POI) is a frequent disorder that affects ~1% of women under 40 years of age. POI, which is characterized by the premature depletion of ovarian follicles and elevated plasma levels of follicle-stimulating hormone (FSH), leads to infertility. Although various etiological factors have been described, including chromosomal abnormalities and gene variants, most cases remain idiopathic. The aim of the present study was to identify and validate functionally new sequence variants in ATG (autophagy-related genes) leading to POI. METHODS: We have reanalyzed, in silico, the exome sequencing data from a previously reported work performed in 69 unrelated POI women. Functional experiments using a classical hallmark of autophagy, the microtubule-associated protein 1 light chain 3ß (LC3), were then used to link these genes to this lysosomal degradation pathway. RESULTS: We venture a functional link between ATG7 and ATG9A variants and POI. We demonstrated that variant ATG7 and ATG9A led to a decrease in autophagosome biosynthesis and consequently to an impairment of autophagy, a key biological process implicated in the preservation of the primordial follicles forming the ovarian reserve. CONCLUSION: Our results unveil that impaired autophagy is a novel pathophysiological mechanism involved in human POI.
Assuntos
Proteína 7 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Proteínas de Membrana/genética , Insuficiência Ovariana Primária/genética , Proteínas de Transporte Vesicular/genética , Adulto , Feminino , Hormônio Foliculoestimulante/genética , Predisposição Genética para Doença , Humanos , Mutação com Perda de Função/genética , Menopausa Precoce/genética , Insuficiência Ovariana Primária/patologia , Sequenciamento do ExomaRESUMO
Mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) are two closely related hormone-activated transcription factors that regulate major pathophysiologic functions. High homology between these receptors accounts for the crossbinding of their corresponding ligands, MR being activated by both aldosterone and cortisol and GR essentially activated by cortisol. Their coexpression and ability to bind similar DNA motifs highlight the need to investigate their respective contributions to overall corticosteroid signaling. Here, we decipher the transcriptional regulatory mechanisms that underlie selective effects of MRs and GRs on shared genomic targets in a human renal cellular model. Kinetic, serial, and sequential chromatin immunoprecipitation approaches were performed on the period circadian protein 1 ( PER1) target gene, providing evidence that both receptors dynamically and cyclically interact at the same target promoter in a specific and distinct transcriptional signature. During this process, both receptors regulate PER1 gene by binding as homo- or heterodimers to the same promoter region. Our results suggest a novel level of MR-GR target gene regulation, which should be considered for a better and integrated understanding of corticosteroid-related pathophysiology.-Le Billan, F., Amazit, L., Bleakley, K., Xue, Q.-Y., Pussard, E., Lhadj, C., Kolkhof, P., Viengchareun, S., Fagart, J., Lombès, M. Corticosteroid receptors adopt distinct cyclical transcriptional signatures.
Assuntos
Regulação da Expressão Gênica , Motivos de Nucleotídeos , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Elementos de Resposta , Transdução de Sinais , Transcrição Gênica , Linhagem Celular , Humanos , Proteínas Circadianas Period/biossíntese , Proteínas Circadianas Period/genética , Receptores de Glucocorticoides/genética , Receptores de Mineralocorticoides/genéticaRESUMO
Mineralocorticoid receptor (MR) mediates the sodium-retaining action of aldosterone in the distal nephron. Herein, we decipher mechanisms by which hypotonicity increases MR expression in renal principal cells. We identify HuR (human antigen R), an mRNA-stabilizing protein, as an important posttranscriptional regulator of MR expression. Hypotonicity triggers a rapid and reversible nuclear export of HuR in renal KC3AC1 cells, as quantified by high-throughput microscopy. We also identify a key hairpin motif in the 3'-untranslated region of MR transcript, pivotal for the interaction with HuR and its stabilizing function. Next, we show that hypotonicity increases MR recruitment onto Sgk1 promoter, a well-known MR target gene, thereby enhancing aldosterone responsiveness. Our data shed new light on the crucial role of HuR as a stabilizing factor for the MR transcript and provide evidence for a short autoregulatory loop in which expression of a nuclear receptor transcriptionally regulating water and sodium balance is controlled by osmotic tone.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Rim/metabolismo , Mineralocorticoides/metabolismo , Pressão Osmótica/fisiologia , Proteínas de Ligação a RNA/metabolismo , Receptores de Mineralocorticoides/metabolismo , Transdução de Sinais/fisiologia , Regiões 3' não Traduzidas/genética , Transporte Ativo do Núcleo Celular/genética , Aldosterona/metabolismo , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Proteínas Imediatamente Precoces/metabolismo , Rim/fisiologia , Osmose/fisiologia , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/genéticaRESUMO
Aldosterone and the Mineralocorticoid Receptor (MR) control hydroelectrolytic homeostasis and alterations of mineralocorticoid signaling pathway are involved in the pathogenesis of numerous human diseases, justifying the need to decipher molecular events controlling MR expression level. Here, we show in renal cells that the RNA-Binding Protein, Human antigen R (HuR), plays a central role in the editing of MR transcript as revealed by a RNA interference strategy. We identify a novel Δ6 MR splice variant, which lacks the entire exon 6, following a HuR-dependent exon skipping event. Using isoform-specific TaqMan probes, we show that Δ6 MR variant is expressed in all MR-expressing tissues and cells and demonstrate that extracelullar tonicity regulates its renal expression. More importantly, this splice variant exerts dominant-negative effects on transcriptional activity of the full-length MR protein. Collectively, our data highlight a crucial role of HuR as a master posttranscriptional regulator of MR expression in response to osmotic stress. We demonstrate that hypotonicity, not only enhances MR mRNA stability, but also decreases expression of the Δ6 MR variant, thus potentiating renal MR signaling. These findings provide compelling evidence for an autoregulatory feedback loop for the control of sodium homeostasis through posttranscriptional events, likely relevant in renal pathophysiological situations.
Assuntos
Processamento Alternativo , Proteína Semelhante a ELAV 1/genética , Rim/metabolismo , Osmorregulação/genética , Receptores de Mineralocorticoides/genética , Sódio na Dieta/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , Proteína Semelhante a ELAV 1/metabolismo , Éxons , Retroalimentação Fisiológica , Furosemida/farmacologia , Homeostase/genética , Humanos , Íntrons , Rim/efeitos dos fármacos , Camundongos , Modelos Moleculares , Concentração Osmolar , Pressão Osmótica , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores de Mineralocorticoides/metabolismo , Sódio na Dieta/administração & dosagem , Homologia Estrutural de Proteína , Privação de Água , Intoxicação por Água/genética , Intoxicação por Água/metabolismo , Intoxicação por Água/fisiopatologiaRESUMO
Nestorone® (NES) is a potent nonandrogenic progestin being developed for contraception. NES is a synthetic progestin that may possess neuroprotective and myelin regenerative potential as added health benefits. In receptor transactivation experiments, NES displayed greater potency than progesterone to transactivate the human progesterone receptor (PR). This was confirmed by docking experiments where NES adopts the same docking position within the PR ligand-binding domain (LBD) as progesterone and forms additional stabilizing contacts between 17α-acetoxy and 16-methylene groups and PR LBD, supporting its higher potency than progesterone. The analog 13-ethyl NES also establishes similar contacts as NES with Met909, leading to comparable potency as NES. In contrast, NES is not stabilized within the human androgen receptor LBD, leading to negligible androgen receptor transactivation. Because progesterone acts in the brain by both PR binding and indirectly via binding of the metabolite allopregnanolone to γ-aminobutyric acid type A receptor (GABAAR), we investigated if NES is metabolized to 3α, 5α-tetrahydronestorone (3α, 5α-THNES) in the brain and if this metabolite could interact with GABAAR. In female mice, low concentrations of reduced NES metabolites were identified by gas chromatography/mass spectrometry in both plasma and brain. Electrophysiological studies showed that 3α, 5α-THNES exhibited only limited activity to enhance GABAAR-evoked responses with WSS-1 cells and did not modulate synaptic GABAARs of mouse cortical neurons. Thus, the inability of reduced metabolite of NES (3α, 5α-THNES) to activate GABAAR suggests that the neuroprotective and myelin regenerative effects of NES are mediated via PR binding and not via its interaction with the GABAAR.
Assuntos
Encéfalo/metabolismo , Anticoncepcionais Femininos/metabolismo , Norprogesteronas/metabolismo , Receptores de GABA-A/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Técnicas de Patch-Clamp , Pregnanolona/metabolismo , Receptores Androgênicos/metabolismo , Receptores de Progesterona/metabolismo , Relação Estrutura-AtividadeRESUMO
Context: Estrogens influence many physiological processes in mammals, including reproduction. Estrogen peripheral actions are mainly mediated through estrogen receptors (ERs) α and ß, encoded by ESR1 and ESR2 genes, respectively. Objective: The study's aim was to describe a family in which 3 members presented with estrogen insensitivity. Design and Setting: Clinical evaluation and genetic and mutational analysis were performed in an academic medical center. Patients and Interventions: An ESR1 mutation was identified in 2 sisters and 1 brother, originating from a consanguineous Algerian family, who did not enter puberty and presented with delayed bone maturation consistent with estrogen insensitivity. The 2 sisters had enlarged multicystic ovaries. Hormonal evaluation as well as genetic and mutational analysis were performed. Results: Hormonal evaluation revealed extremely high plasma 17ß-estradiol (>50-fold normal range) associated with elevated gonadotropin levels (greater than threefold normal range), highly suggestive of estrogen resistance. The 3 affected patients carried a homozygous mutation of a highly conserved arginine 394 for which histidine was substituted through an autosomal recessive mode of transmission. Structural and functional analysis of the mutant ERα revealed strongly reduced transcriptional activity and the inability to securely anchor the activating hormone, estradiol, compared with wild-type ERα. A group of other potential ER activating ligands were tested, but none overcame the estrogen insensitivity in these patients. Conclusion: Description and analysis of this family of patients with mutant ERα provide additional clinical findings toward identification and characterization of what was previously thought to be a highly rare clinical condition.
Assuntos
Resistência a Medicamentos/genética , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Mutação/genética , Maturidade Sexual/genética , Adolescente , Adulto , Biomarcadores/análise , Feminino , Seguimentos , Humanos , Masculino , Linhagem , Prognóstico , Ligação Proteica , Ativação Transcricional , Adulto JovemRESUMO
CONTEXT: Idiopathic primary ovarian insufficiency (POI) is a major cause of amenorrhea and infertility. POI affects 1% of women before age 40 years, and several genetic causes have been reported. To date, POI has been considered a monogenic disorder. OBJECTIVE: The aim of this study was to identify novel gene variations and to investigate if individuals with POI harbor mutation in multiple loci. PATIENTS AND METHODS: One hundred well-phenotyped POI patients were systematically screened for variants in 19 known POI loci (and potential candidate genes) using next-generation sequencing. RESULTS: At least one rare protein-altering gene variant was identified in 19 patients, including missense mutations in new candidate genes, namely SMC1ß and REC8 (involved in the cohesin complex) and LHX8, a gene encoding a transcription factor. Novel or recurrent deleterious mutations were also detected in the known POI candidate genes NOBOX, FOXL2, SOHLH1, FIGLA, GDF9, BMP15, and GALT. Seven patients harbor mutations in two loci, and this digenicity seems to influence the age of symptom onset. CONCLUSIONS: Genetic anomalies in women with POI are more frequent than previously believed. Digenic findings in several cases suggest that POI is not a purely monogenic disorder and points to a role of digenicity. The genotype-phenotype correlations in some kindreds suggest that a synergistic effect of several mutations may underlie the POI phenotype.
Assuntos
Insuficiência Ovariana Primária/genética , Adolescente , Adulto , Feminino , Loci Gênicos , Genótipo , Humanos , Mutação , Fenótipo , Análise de Sequência de DNA , Adulto JovemRESUMO
Generalized glucocorticoid resistance is associated with glucocorticoid receptor (GR; NR3C1) mutations. Three novel heterozygous missense NR3C1 mutations (R477S, Y478C, and L672P) were identified in patients presenting with adrenal incidentalomas, glucocorticoid excess without Cushing syndrome. Dexamethasone (DXM) binding studies demonstrated that the affinity of GRR477S and GRY478C mutants, located in the DNA-binding domain (DBD) of GR, was similar to wild-type GR (Kd = 2-3 nM). In contrast, GRL672P mutant, located in the ligand-binding domain (LBD) of GR, was unable to bind glucocorticoids and was more sensitive to protein degradation. GR subcellular distribution revealed a marked decrease in DXM-induced nuclear translocation of GRR477S and GRY478C mutants, whereas GRL672P remained exclusively cytoplasmic. Chromatin immunoprecipitation demonstrated impaired recruitment of DBD mutants onto the regulatory sequence of FKBP5. Transactivation assays disclosed the lack of transcriptional activity of GRR477S and GRL672P , whereas GRY478C had a reduced transactivation capacity. Three-dimensional modeling indicated that R477S lost two essential hydrogen bonds with DNA, Y478C resulted in altered interaction with surrounding amino-acids, destabilizing DBD, whereas L672P altered the H8 helix folding, leading to unstructured LBD. This study identifies novel NR3C1 mutations with their molecular consequences on altered GR signaling and suggests that genetic screening of NR3C1 should be conducted in patients with subclinical hypercorticism.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Resistencia a Medicamentos Antineoplásicos , Glucocorticoides/farmacologia , Mutação Puntual , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Neoplasias das Glândulas Suprarrenais/tratamento farmacológico , Adulto , Animais , Sítios de Ligação , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Síndrome de Cushing/genética , Citoplasma/metabolismo , DNA de Neoplasias/metabolismo , Dexametasona/farmacologia , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Transporte Proteico , Receptores de Glucocorticoides/metabolismoRESUMO
Breast cancer is a hormone-dependent disease in which estrogen signaling targeting drugs fail in about 10 % due to resistance. Strong evidences highlighted the mitogen role of progesterone, its ligands, and the corresponding progesterone receptor (PR) isoforms in mammary carcinoma. Several PR antagonists have been synthesized; however, some of them are non-selective and led to side or toxic effects. Herein, we evaluated the anti-tumor activity of a commercially available PR modulator, ulipristal acetate (UPA), and a new selective and passive PR antagonist "APR19" in a novel preclinical approach based on patient-derived breast tumor (HBCx-34) xenografted in nude mice. As opposed to P4 that slightly reduces tumor volume, UPA and APR19 treatment for 42 days led to a significant 30 % reduction in tumor weight, accompanied by a significant 40 % retardation in tumor growth upon UPA exposure while a 1.5-fold increase in necrotic areas was observed in APR19-treated tumors. Interestingly, PR expression was upregulated by a 2.5-fold factor in UPA-treated tumors while APR19 significantly reduced expression of both PR and estrogen receptor α, indicating a potential distinct molecular mechanism among PR antagonists. Cell proliferation was clearly reduced in UPA group compared to vehicle conditions, as revealed by the significant reduction in Ki-67, Cyclin D1, and proliferating cell nuclear antigen (PCNA) expression. Likewise, an increase in activated, cleaved poly(ADP-ribose) polymerase (PARP) expression was also demonstrated upon UPA exposure. Collectively, our findings provide direct in vivo evidence for anti-progestin-mediated control of human breast cancer growth, given their anti-proliferative and pro-apoptotic activities, supporting a potential role in breast cancer therapy.
Assuntos
Androstenos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Homosteroides/administração & dosagem , Norpregnadienos/administração & dosagem , Receptores de Progesterona/metabolismo , Androstenos/farmacologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Homosteroides/farmacologia , Humanos , Camundongos , Camundongos Nus , Norpregnadienos/farmacologia , Receptores de Progesterona/genética , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aldosterone regulates sodium homeostasis by activating the mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily. Hyperaldosteronism leads todeleterious effects on the kidney, blood vessels, and heart. Although steroidal antagonists such as spironolactone and eplerenone are clinically useful for the treatment of cardiovascular diseases, they are associated with several side effects. Finerenone, a novel nonsteroidal MR antagonist, is presently being evaluated in two clinical phase IIb trials. Here, we characterized the molecular mechanisms of action of finerenone and spironolactone at several key steps of the MR signaling pathway. Molecular modeling and mutagenesis approaches allowed identification of Ser-810 and Ala-773 as key residues for the high MR selectivity of finerenone. Moreover, we showed that, in contrast to spironolactone, which activates the S810L mutant MR responsible for a severe form of early onset hypertension, finerenone displays strict antagonistic properties. Aldosterone-dependent phosphorylation and degradation of MR are inhibited by both finerenone and spironolactone. However, automated quantification of MR subcellular distribution demonstrated that finerenone delays aldosterone-induced nuclear accumulation of MR more efficiently than spironolactone. Finally, chromatin immunoprecipitation assays revealed that, as opposed to spironolactone, finerenone inhibits MR, steroid receptor coactivator-1, and RNA polymerase II binding at the regulatory sequence of the SCNN1A gene and also remarkably reduces basal MR and steroid receptor coactivator-1 recruitment, unraveling a specific and unrecognized inactivating mechanism on MR signaling. Overall, our data demonstrate that the highly potent and selective MR antagonist finerenone specifically impairs several critical steps of the MR signaling pathway and therefore represents a promising new generation MR antagonist.
Assuntos
Aldosterona/farmacologia , Naftiridinas/farmacologia , Coativador 1 de Receptor Nuclear/metabolismo , Receptores de Mineralocorticoides/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Western Blotting , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Canais Epiteliais de Sódio/genética , Células HEK293 , Humanos , Cinética , Microscopia de Fluorescência , Mutação , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Receptores de Mineralocorticoides/genética , Transdução de Sinais/efeitos dos fármacos , Espironolactona/farmacologia , Ativação Transcricional/efeitos dos fármacosRESUMO
Aldosterone exerts its effects mainly by activating the mineralocorticoid receptor (MR), a transcription factor that regulates gene expression through complex and dynamic interactions with coregulators and transcriptional machinery, leading to fine-tuned control of vectorial ionic transport in the distal nephron. To identify genome-wide aldosterone-regulated MR targets in human renal cells, we set up a chromatin immunoprecipitation (ChIP) assay by using a specific anti-MR antibody in a differentiated human renal cell line expressing green fluorescent protein (GFP)-MR. This approach, coupled with high-throughput sequencing, allowed identification of 974 genomic MR targets. Computational analysis identified an MR response element (MRE) including single or multiple half-sites and palindromic motifs in which the AGtACAgxatGTtCt sequence was the most prevalent motif. Most genomic MR-binding sites (MBSs) are located >10 kb from the transcriptional start sites of target genes (84%). Specific aldosterone-induced recruitment of MR on the first most relevant genomic sequences was further validated by ChIP-quantitative (q)PCR and correlated with concomitant and positive aldosterone-activated transcriptional regulation of the corresponding gene, as assayed by RT-qPCR. It was notable that most MBSs lacked MREs but harbored DNA recognition motifs for other transcription factors (FOX, EGR1, AP1, PAX5) suggesting functional interaction. This work provides new insights into aldosterone MR-mediated renal signaling and opens relevant perspectives for mineralocorticoid-related pathophysiology.
Assuntos
Aldosterona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Rim/metabolismo , Receptores de Mineralocorticoides/biossíntese , Elementos de Resposta/fisiologia , Transdução de Sinais/efeitos dos fármacos , Aldosterona/metabolismo , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Humanos , Rim/citologia , Receptores de Mineralocorticoides/genética , Transdução de Sinais/fisiologiaRESUMO
There is a therapeutic need for glucocorticoid receptor (GR) ligands that distinguish between the transrepression and transactivation activity of the GR, the later thought to be responsible for side effects. These ligands are known as "dissociated glucocorticoids" (dGCs). The first published dGCs, RU24782 (9α-fluoro-11ß-hydroxy-16α-methylpregna-21-thiomethyl-1,4-diene-3,20-dione) and RU24858 (9α-fluoro-11ß-hydroxy-16α-methylpregna-21-cyanide-1,4-diene-3,20-dione), do not have the 17α-hydroxyl group that characterizes dexamethasone (Dex; 9α-fluoro-11ß,17α,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione), and they differ from one another by having C21-thiomethyl and C21-cyanide moieties, respectively. Our aim was therefore to establish the structural basis of their activity. Both RU24782 and RU24858 induced a transactivation activity highly dependent on the GR expression level but always lower than dexamethasone. They also display less ability than dexamethasone to trigger steroid receptor coactivator 1 (SRC-1) recruitment and histone H3 acetylation. Docking studies, validated by mutagenesis experiments, revealed that dGCs are not anchored by Gln642, in contrast to Dex, which is hydrogen bonded to this residue via its 17α-hydroxyl group. This contact is essential for SRC-1 recruitment and subsequent dexamethasone-induced GR transactivation, but not transrepression. The ability of dGCs to make contacts with Ile747, for both RU24858 and RU24782 and with Asn564 for RU24858 are not strong enough to maintain GR in a conformation able to efficiently recruit SRC-1, unless SRC-1 is overexpressed. Overall, our findings provide some structural guidelines for the synthesis of potential new dissociated glucocorticoids with a better therapeutic ratio.
Assuntos
Glucocorticoides/farmacologia , Receptores de Glucocorticoides/genética , Ativação Transcricional/efeitos dos fármacos , Transporte Ativo do Núcleo Celular , Animais , Sítios de Ligação , Células COS , Células Cultivadas , Chlorocebus aethiops , Dexametasona/farmacologia , Glucocorticoides/química , Glucocorticoides/metabolismo , Humanos , Coativador 1 de Receptor Nuclear/fisiologia , Regiões Promotoras Genéticas , Conformação Proteica , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/genéticaRESUMO
Currently available progesterone (P4) receptor (PR) antagonists, such as mifepristone (RU486), lack specificity and display partial agonist properties, leading to potential drawbacks in their clinical use. Recent x-ray crystallographic studies have identified key contacts involved in the binding of agonists and antagonists with PR opening the way for a new rational strategy for inactivating PR. We report here the synthesis and characterization of a novel class of PR antagonists (APRn) designed from such studies. The lead molecule, the homosteroid APR19, displays in vivo endometrial anti-P4 activity. APR19 inhibits P4-induced PR recruitment and transactivation from synthetic and endogenous gene promoters. Importantly, it exhibits high PR selectivity with respect to other steroid hormone receptors and is devoid of any partial agonist activity on PR target gene transcription. Two-hybrid and immunostaining experiments reveal that APR19-bound PR is unable to interact with either steroid receptor coactivators 1 and 2 (SRC1 and SCR2) or nuclear receptor corepressor (NcoR) and silencing mediator of retinoid acid and thyroid hormone receptor (SMRT), in contrast to RU486-PR complexes. APR19 also inhibits agonist-induced phosphorylation of serine 294 regulating PR transcriptional activity and turnover kinetics. In silico docking studies based on the crystal structure of the PR ligand-binding domain show that, in contrast to P4, APR19 does not establish stabilizing hydrogen bonds with the ligand-binding cavity, resulting in an unstable ligand-receptor complex. Altogether, these properties highly distinguish APR19 from RU486 and likely its derivatives, suggesting that it belongs to a new class of pure antiprogestins that inactivate PR by a passive mechanism. These specific PR antagonists open new perspectives for long-term hormonal therapy.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Homosteroides/farmacologia , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Receptores de Progesterona/antagonistas & inibidores , Esteroides/farmacologia , Transporte Ativo do Núcleo Celular , Androstenos , Sítios de Ligação , Linhagem Celular Tumoral/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HEK293 , Homosteroides/síntese química , Humanos , Modelos Moleculares , Ligação Proteica , Proteólise/efeitos dos fármacos , Receptores de Progesterona/agonistas , Receptores de Progesterona/metabolismo , Esteroides/síntese química , Fatores de Transcrição/metabolismoRESUMO
CONTEXT: KISS1R mutations have been reported in few patients with normosmic congenital hypogonadotropic hypogonadism (nCHH) (OMIM #146110). OBJECTIVE: To describe in detail nCHH patients with biallelic KISS1R mutations belonging to 2 unrelated families, and to functionally characterize a novel KISS1R mutation. RESULTS: An original mutant, p.Tyr313His, was found in the homozygous state in 3 affected kindred (2 females and 1 male) from a consanguineous Portuguese family. This mutation, located in the seventh transmembrane domain, affects a highly conserved amino acid, perturbs the conformation of the transmembrane segment, and impairs MAP kinase signaling and intracellular calcium release. In the second family, a French Caucasian male patient with nCHH was found to carry two recurrent mutations in the compound heterozygous state (p.Leu102Pro/Stop399Arg). In this man, pulsatile GnRH (Gonadotropin Releasing Hormone) administration restored pulsatile LH (Luteinizing Hormone) secretion and testicular hormone secretion. Later, long-term combined gonadotropin therapy induced spermatogenesis, enabling 3 successive pregnancies that resulted in 2 miscarriages and the birth of a healthy boy. CONCLUSION: We show that a novel loss-of-function mutation (p.Tyr313His) in the KISS1R gene can cause familial nCHH, revealing the crucial role of this amino acid in KISS1R function. The observed restoration of gonadotropin secretion by exogenous GnRH administration further supports, in humans, the hypothalamic origin of the gonadotropin deficiency in this genetic form of nCHH.
Assuntos
Predisposição Genética para Doença/genética , Hipogonadismo/genética , Mutação , Receptores Acoplados a Proteínas G/genética , Alelos , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Cálcio/metabolismo , Chlorocebus aethiops , Análise Mutacional de DNA , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Saúde da Família , Feminino , Células HEK293 , Humanos , Hipogonadismo/congênito , Kisspeptinas/farmacologia , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Fosforilação/efeitos dos fármacos , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Receptores de Kisspeptina-1 , Homologia de Sequência de AminoácidosRESUMO
Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by mutations in the HEXB gene encoding the beta subunit of hexosaminidases A and B, two enzymes involved in GM2 ganglioside degradation. Eleven French Sandhoff patients with infantile or juvenile forms of the disease were completely characterized using sequencing of the HEXB gene. A specific procedure was developed to facilitate the detection of the common 5'-end 16kb deletion which was frequent (36% of the alleles) in our study. Eleven other disease-causing mutations were found, among which four have previously been reported (c.850C>T, c.793T>G, c.115del and c.800_817del). Seven mutations were completely novel and were analyzed using molecular modelling. Two deletions (c.176del and c.1058_1060del), a duplication (c.1485_1487dup) and a nonsense mutation (c.552T>G) were predicted to strongly alter the enzyme spatial organization. The splice mutation c.558+5G>A affecting the intron 4 consensus splice site led to a skipping of exon 4 and to a truncated protein (p.191X). Two missense mutations were found among the patients studied. The c.448A>C mutation was probably a severe mutation as it was present in association with the known c.793T>G in an infantile form of Sandhoff disease and as it significantly modified the N-terminal domain structure of the protein. The c.171G>C mutation resulting in a p.W57C amino acid substitution in the N-terminal region is probably less drastic than the other abnormalities as it was present in a juvenile patient in association with the c.176del. Finally, this study reports a rapid detection of the Sandhoff disease-causing alleles facilitating genetic counselling and prenatal diagnosis in at-risk families.
Assuntos
Alelos , Mutação , Doença de Sandhoff/genética , Cadeia beta da beta-Hexosaminidase/genética , Substituição de Aminoácidos , Feminino , França , Humanos , Lactente , Recém-Nascido , Íntrons , Masculino , Sítios de Splice de RNA , Doença de Sandhoff/diagnósticoRESUMO
The first and critical step in the mechanism of aldosterone action is its binding to the mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily. Over the last 40 years, numerous studies have attempted to determine the structural determinants of ligand-binding to MR. An initial set of data showed that hsp90 is bound to the receptor via specific regions and maintains it in a ligand-binding competent state. Site-directed mutagenesis and functional studies guided by a 3D model of the MR ligand-binding domain (LBD) made it possible to identify the residues responsible for the high affinity and selectivity for aldosterone, and to characterize the mechanisms of MR activation and inactivation. The recent determination of the X-ray crystal structures of the LBD of the wild-type MR and MR(S810L), which is responsible for a familial form of hypertension, has made it possible to elucidate the peculiar mechanism of activation of MR(S810L) and established a clear structure/activity relationship for steroidal and non-steroidal MR antagonists.